Micropropagation of apple — A review

Micropropagation of apple has played an important role in the production of healthy, disease-free plants and in the rapid multiplication of scions and rootstocks with desirable traits. During the last few decades, in apple, many reliable methods have been developed for both rootstocks and scions from a practical, commercial point of view. Successful micropropagation of apple using pre-existing meristems (culture of apical buds or nodal segments) is influenced by several internal and external factors including ex vitro (e.g. genotype and physiological state) and in vitro conditions (e.g., media constituents and light). Specific requirements during stages of micropropagation, such as the establishment of in vitro cultures, shoot multiplication, rooting of microshoots and acclimatization are summarized in this review. New approaches for increasing shoot multiplication and rooting for apple and current use of micropropagated plantlets as tools in basic and applied research are also discussed.     

Detoxification of aflatoxin B1 by an aqueous extract from leaves of Adhatoda vasica Nees

The effectiveness of aqueous extracts of various medicinal plants in detoxification of aflatoxin B1 (AFB1) was tested in vitro by thin-layer chromatography and enzyme-linked immunosorbent assay (ELISA). Among the different plant extracts, the leaf extract of Vasaka (Adhatoda vasica Nees) showed the maximum degradation of AFB1 (≥98%) after incubation for 24 h at 37 °C. The aflatoxin detoxifying activity of the A. vasica leaf extract was significantly reduced by heating to 100 °C for 10 min or autoclaving at 121 °C for 20 min. Dialysis had no effect on aflatoxin detoxifying ability of A. vasica extract and the dialyzed extract showed similar level of detoxification of AFB1 as that of the untreated extract. A time course study of aflatoxin detoxification by A. vasica extract showed that 69% of the toxin was degraded within 6 h and ≥95% degradation was observed after 24 h of incubation. Detoxification of AFB1 by A. vasica extract was further confirmed by liquid chromatography–mass spectrometry (LC–MS) analysis. Phytochemical analysis revealed the presence of alkaloids in methanolic extract of A. vasica leaves. A partially purified alkaloid from A. vasica leaves by preparative TLC exhibited strong AFB1 detoxification activity.     

Endogenous isoflavone methylation correlates with the in vitro rooting phases of Spartium junceum L. (Leguminosae)

Spartium junceum L. (Leguminosae) is a perennial shrub, native to the Mediterranean region in southern Europe, widespread in all the Italian regions and, as a leguminous species, it has a high isoflavone content. An in vitro culture protocol was developed for this species starting from stem nodal sections of in vivo plants, and isoflavone components of the in vitro cultured tissues were studied by means of High Performance Liquid Chromatography (HPLC) analytical techniques. Two main isoflavones were detected in the S. junceum tissues during the in vitro propagation phases: Genistein (4′,5,7-Trihydroxyisoflavone), already reported in this species, and its methylated form 4′,5,7-Trimethoxyisoflavone, detected for the first time in this plant species (0.750 ± 0.02 mg g⁻¹ dry tissue). The presence of both of these compounds in S. junceum tissues was consistently detected during the in vitro multiplication phase. The absence of the methylated form within plant tissues in the early phases of the in vitro adventitious root formation was correlated with its negative effect displayed on root induction and initiation phases, while its presence in the final “root manifestation” phase influenced positively the rooting process. The unmethylated form, although detectable in tissues in the precocious rooting phases, was no longer present in the final rooting phase. Its effect on rooting, however, proved always to be beneficial.     

Micropropagation of Sterculia urens Roxb. — an endangered tree species

An in vitro procedure for large scale multiplication of Sterculia urens Roxb. (Gum Kadaya Tree) has been developed using cotyledonary node segments. An average of 4.0 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 2.0 mgl^–1 6-benzyl amino-purine (BAP) within 21 days of initial culture. Upon subsequent subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots were successfully rooted. Rooted plantlets were transferred to plastic pots containing soil under mist house conditions before they were finally exposed to an external environment. Fifty seven per cent of the plantlets survived in nursery sheds.     

In vitro regeneration of adult Pinus sylvestris L. trees

The propagation of adult conifer trees by tissue culture has been studied for the last twenty years, but problems related to the juvenile to adult phase change of trees have limited the practical applications of these tissue culture procedures. This paper describes a micropropagation protocol for the in vitro propagation of mature Scots pine trees. In this study, dormant shoot buds, which had not started to elongate, were collected from twenty-one adult Pinus sylvestris trees (> 15 years old) during the winter. The sampled buds were cut transversely into slices of 0.5 to 1 cm in thickness and were cultured on three types of culture media (DCR, WP and LPm) supplemented with four cytokinins (BA, mT, Tdz and Z), at two different concentrations (25 and 50 µM), except for Tdz, whose concentrations were diluted to 5 µM and 2.5 µM. The evaluated culture media did not show significant differences in the bud organogenesis capacity. In fact, the highest organogenic response was obtained with buds cultured on DCR and WP media and by explants cultured on medium supplemented with 25 µM meta-topolin. This protocol is a successful and efficient biotechnological approach to the micropropagation of adult P. sylvestris trees.     

In vitro propagation ofBoswellia serrata Roxb

Anin vitro procedure for large scale multiplication ofBoswellia serrata Roxb. has been developed using cotyledonary node segments. In average 4 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ napthaleneacetic acid (NAA) within 22 d. By repeated subculture technique 90–100 shoots per node could be obtained after 88 d of initial culture. Shoots could be rooted on MS medium containing 1/4 salts, 1% saccharose, and a combination of 0.5 mg dm⁻³ indole-3-butyric acid (IBA) and 0.25 mg dm⁻³ indole-3-acetic acid (IAA). Addition of antioxidants like polyvinylpyrrolidone (PVP-50 mg dm⁻³) and ascorbic acid (100 mg dm⁻³) in both multiplication and rooting media prevented browning of cultures. Approximately 80% of shoots rooted within 8–10 d. Rooted plantlets were kept for 15 d in culture bottles containing Soilrite^TM irrigated with a nutrient solution containing 1/4 MS salts and finally transferred to pots containing soil: Soilrite^TM (1∶1), mixture with 70% transplantation success.     

Rapid Axillary Bud Proliferation and ex vitro Rooting of Eupatorium triplinerve

Effective protocol was established for micropropagation of the medicinal plant Eupatorium triplinerve Vahl through rapid axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium fortified with 8.87 M benzylaminopurine (BAP) and 2.46 M indole-3-butyric acid (IBA) was the best for axillary bud proliferation and developed a mean of 8.1 shoots per node. Excision and culture of the node segments of the in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 d. Shoot multiplication did not exhibit decrease in the number of shoots even at 7^th subculture. Dipping of the basal end of shoots in 2.46 M IBA solution for 10 d induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100 %). Ex vitro rooting by direct transfer of the shoots from multiplication medium showed 92 % survival.     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon

Brachypodium distachyon (purple false brome) is a herbaceous species belonging to the grass subfamily Pooideae, which also includes major crops like wheat, barley, oat and rye. The species has been established as experimental model organism for understanding and improving cereal crops and temperate grasses. The complete genome of Bd21, the community standard line of B. distachyon, has been sequenced and protocols for Agrobacterium-mediated transformation have been published. Further improvements to the experimental platform including better evaluation systems for transgenic plants are still needed. Here we describe the growth conditions for Bd21 plants yielding highly responsive immature embryos that can generate embryogenic calli for transformation. A prolonged 20-h photoperiod produced seeds with superior immature embryos. In addition, osmotic treatment of embryogenic calli enhanced the efficiency of transfection by particle bombardment. We generated transgenic plants expressing Arabidopsis thaliana galactinol synthase 2 (AtGolS2) in these experiments. AtGolS2-expressing transgenics displayed significantly improved drought tolerance, increasing with increased expression of AtGolS2. These results demonstrate that AtGolS2 can confer drought tolerance to monocots and confirm that Brachypodium is a useful model to further explore ways to understand and improve major monocot crop species.     

Spontaneous sexual differentiation of Mesocestoides corti tetrathyridia in vitro

Barrett N. J., Smyth J. D. and Ong S. J. 1982. Spontaneous sexual differentiation of Mesocestoides corti tetrathyridia in vitro. International Journal for Parasitology12: 315–322. Tetrathyridia of Mesocestoides corti, from the body cavity of mice, maintained in the laboratory by intraperitoneal infection, were used for in vitro culture. In an initial experiment, after 50 days asexual multiplication in vitro one tetrathyridium spontaneously segmented and developed into a sexually mature adult. Further experiments were carried out in an attempt to determine the conditions favouring segmentation and sexual differentiation. A combination of 5 or 10 ml liquid medium S1OE.H (basically composed of CMRL 1066 and foetal calf serum with supplements) changed every 3 days, in a Leighton tube (19 × 105 mm), rotated at 38°C and gassed with 10 or 20% CO₂, containing between 100 and 200 tetrathyridia, has proved to be most suitable so far. Numerous adult worms with normal male and female genitalia have been obtained in this system. However, segmentation is sporadic, rather than consistent and only a few shelled eggs with hooked oncospheres have so far been obtained, suggesting that impregnation and fertilization in vitro is not fully comparable with that in vivo.     

Cryptosporidium: New developments in cell culture

Studies on Cryptosporidium species have been hampered by the limited amount of parasitic stages available for research. One of the major objectives of many laboratories is to develop a reproducible culture model for this important parasite. Recent research has resulted in long-term culturing of Cryptosporidium in cell culture using pH modification, sub-culturing and gamma irradiation. Further advances in the in vitro culturing of Cryptosporidium revealed that this parasite can complete its life cycle in culture medium overcoming the problem of using the host cells, as host cell overgrowth and aging resulted in the termination of the Cryptosporidium life cycle prior to its completion. Improved methods for visualizing life cycle stages in cell-free culture have also been developed. This review will discuss factors that can influence the success of Cryptosporidium culture in vitro and propose new ideas for the future optimization of the cell-free culture system.     

In vitro culture of lavenders (Lavandula spp.) and the production of secondary metabolites

Lavenders (Lavandula spp., Lamiaceae) are aromatic ornamental plants that are used widely in the food, perfume and pharmaceutical industries. The large-scale production of lavenders requires efficient in vitro propagation techniques to avoid the overexploitation of natural populations and to allow the application of biotechnology-based approaches for plant improvement and the production of valuable secondary metabolites. In this review we discuss micropropagation methods that have been developed in several lavender species, mainly based on meristem proliferation and organogenesis. Specific requirements during stages of micropropagation (establishment, shoot multiplication, root induction and acclimatization) and requisites for plant regeneration trough organogenesis, as an important step for the implementation of plant improvement programs, were revised. We also discuss different methods for the in vitro production of valuable secondary metabolites, focusing on the prospects for highly scalable cultures to meet the market demand for lavender-derived products.     

Propagation of an elite high biomass-producing genotype of Atriplex canescens by axillary enhancement

A technique for rapid in vitro multiplication and rooting of Atriplex canescens microshoots and establishment of plantlets in the soil is described. The influence of growth regulators in the multiplication medium on shoot growth, rooting, and soil establishment of in vitro-derived plants is assessed. Rapid propagation was achieved by the culture of shoot tips or lateral buds on a medium supplemented with 1 mg per liter of benzyladenine. Shoots were rooted in vitro on a medium supplemented with 1 mg per liter indolebutyric acid prior to placement in the soil. Plants were established successfully in the greenhouse through the maintenance of a high humidity environment for the first 21 days and were subsequently transferred to the greenhouse and field.     

Pathogenicity of Pochonia species on eggs of Meloidogyne javanica

Among fungi, species of the genus Pochonia Batista & O.M. Fonseca are considered as promising biological control agents with high potential to reduce root-knot nematode (RKN) and nematode populations. In this research we investigated Fars province of Iran for the presence of Pochonia spp., compared pathogenicity of different Pochonia species on eggs of RKN in vitro, and selected the best isolates for further studies. During 2004-2006, 128 soil samples of fields infested with cyst nematodes and 18 soil samples infested with RKN were collected from Fars province of Iran. In vitro pathogenicity tests were carried out on 36 isolates of Pochonia spp. obtained from CBS and IRAN culture collections. The seven best isolates of this experiment were selected for greenhouse test and their ability in controlling RKN was examined in natural soil. In greenhouse test fresh weight of plant’s tops and roots, gall index, nematode multiplication, second-stage juveniles’ population in soil, reproduction rate (P_f/P_i), proportion of infected eggs, control efficacy, root colonization and soil colony forming units were determined. In vitro pathogenicity of Pochonia on RKN eggs varied between 39% and 95% eggs infected. In greenhouse experiment, three isolates are promising for control of RKN and selected isolates are subjected to more extensive testing to determine their effectiveness in a range of conditions before being developed as commercial biological control agents.     

Salvianolic acid B inhibits myofibroblast transdifferentiation in experimental pulmonary fibrosis via the up-regulation of Nrf2

Salvianolic acid B (SalB) is one of the most bioactive components extracted from Salvia miltiorrhiza, and its antioxidant capacity corresponds with its protective effects against cell injury from oxidative stress. The aim of the present study was to evaluate the effect of SalB on experimental pulmonary fibrosis and its ability to ameliorate the oxidative/antioxidative imbalance during fibrosis pathogenesis. The anti-fibrotic activity of SalB was first confirmed in Transforming growth factor β1(TGF-β1)-stimulated MRC-5 cells. The protection of SalB against oxidative stress during fibrogenesis in vitro was verified by detecting ROS production, the levels of glutathione (GSH) and malondialdehyde (MDA). The Western blot and PCR results indicated that SalB could up-regulate nuclear factor erythroid-derived 2-like 2 (Nrf2) at both the protein and mRNA levels and induce Nrf2 nuclear translocation in vitro, which may be the mechanism underlying the anti-fibrotic capacity of SalB. Furthermore, the anti-fibrotic and antioxidant capacities of SalB in vivo were confirmed in rats with BLM-induced pulmonary fibrosis. The immunohistochemistry results showed that Nrf2 was absent in fibroblastic foci (FF) areas, while the SalB treatment could increase the expression of Nrf2 in lung tissues, especially in FF areas.     

Plasmodium falciparum: Antimalarial activity in culture of sinefungin and other methylation inhibitors

A culture line of Plasmodium falciparum (FCR-3/Gambia) was exposed in vitro for a 2-day period to several analogs of adenosylhomocysteine. Minimal concentrations giving complete inhibition of growth were 0.2 mM for 3-deazaadenosine, 0.2 mM for 5′-deoxy-5′-(isobutylthio)-3-deazaadenosine, and 0.3 μM for sinefungin. The effects of the first two of these compounds were potentiated by homocysteine-thiolactone, suggesting that they were inhibiting methylation reaction(s) indirectly via adenosylhomocysteine hydrolase (EC 3.3.1.1).     

Micropropagation of Three Varieties of Caralluma adscendens via Nodal Explants

A procedure for in vitro propagation of pharmaceutically valuable varieties of Caralluma adscendens from nodal explant, is described. The highest shoot multiplication with 80% frequency was achieved within one month on Murashige and Skoog’s medium supplemented with 8.87 μM BA. Shoot multiplication occurred in subsequent subcultures in culture bottles on MS medium. Regenerated shoots were rooted on half strength MS medium supplemented with NAA (0.54 μM) in all the three varieties. The rooted plants were hardened for establishment in soil.